异嗪皮啶对人乳腺癌干细胞凋亡相关基因Bcl-2与Caspase家族表达的影响

目的: 研究草珊瑚中异嗪皮啶成分对乳腺癌干细胞中凋亡相关基因通路Bcl-2、Caspase-3、Caspase-8基因表达影响，进而明确其抑制增殖、迁移和促凋亡的机制。方法: 无血清培养法诱导MDA-MB-231细胞株富集乳腺癌干细胞，流式细胞仪分选CD44⁺ /CD24 ^-/low干细胞群。各组分别给予0、17、50、150、450 μmol/L异嗪皮啶，CCK-8法观察给药后24、48、72 h的细胞活力；细胞划痕实验法检测给药后细胞痕道宽度变化值并判断其对细胞迁移能力的影响；Annexin V-FITC/PI染色法检测给药后细胞总凋亡率变化；实时荧光定量PCR测定各组Bcl-2、Caspase-3、Caspase-8基因的mRNA水平；Western blot检测上述基因的蛋白水平。结果: 与对照组比较，50、150和450 μmol/L异嗪皮啶组24、48、72 h的细胞活力降低，细胞迁移能力下降。与对照组比较，50、150和450 μmol/L异嗪皮啶组细胞总凋亡率高于对照组，Bcl-2基因mRNA和蛋白表达水平降低，Caspase-3和Caspase-8基因mRNA和蛋白表达水平升高。以上结果均呈明显剂量效应关系。结论: 异嗪皮啶能通过下调Bcl-2基因表达与激活Caspase基因家族诱导乳腺癌干细胞的凋亡，同时能抑制其增殖与迁移。     

Characterization of Apoptosis Induced by Emodin and Related Regulatory Mechanisms in Human Neuroblastoma Cells

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10–20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10–20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10–20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²⁺, NO, p53, caspase-9 and caspase-3.     

Peptides from the N-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC50 value of this peptide was 6.3 micrograms/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC50 values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.     

小鼠脾淋巴细胞辐射死亡与凋记之间的关系

探讨细胞凋亡在小鼠脾脏辐射损伤与重建中的作用及其机制,为急性放射病的防治提供依据。用原位末端标记、ＤＮＡ电泳和免疫组化技术观察经２￣８Ｇｙ不同剂量γ射线整体照射后,小鼠脾脏淋巴细胞凋亡的动态变化过程和Ｂａｘ、Ｂｃｌ－２蛋白在其中的作用。结果表明,（１）照射后早期淋巴细胞凋亡率迅速增加,如６Ｇｙ照射后４ｈ和１ｄ凋亡率分别为对照值的４．９和９．７倍;凋亡率还随照射剂量的增加而迅速升高;照射后８ｈ当照射     

Optimal modification of annexin V with fluorescent dyes

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N-hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.     

Killing Me Softly—Future Challenges in Apoptosis Research

The induction of apoptosis, a highly regulated and clearly defined mode of cell dying, is a vital tenet of modern cancer therapy. In this review we focus on three aspects of apoptosis research which we believe are the most crucial and most exciting areas currently investigated and that will need to be better understood in order to enhance the efficacy of therapeutic measures. First, we discuss which target to select for cancer therapy and argue that not the cancer cell as such, but its interaction with the microenvironment is a more promising and genetically stable site of attack. Second, the complexity of combination therapy is elucidated using the PI3-K-mediated signaling network as a specific example. Here we show that the current clinical approach to sensitize malignancies to apoptosis by maximal, prolonged inhibition of so-called survival pathways can actually be counter productive. Third, we propose that under certain conditions which will need to be clearly defined in future, chronification of a tumor might be preferable to the attempt at a cure. Finally, we discuss further problems with utilizing apoptosis induction in cancer therapy and propose a novel potential therapeutic approach that combines the previously discussed features.     

Regulation of TRAIL-Receptor Expression by the Ubiquitin-Proteasome System

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor (TRAIL-R) family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC), caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS) has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB) activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients.     

Multiple‐valued immune network with apoptosis system

In this paper, we describe a new model of immune network based on biological immune response network. We propose an immunity like multiple‐valued network with apoptosis mechanism. The model is based on the interaction between B cells and T cells and the biological apoptosis mechanism in the human body. With the mechanism, a naturally immune system can be reproduced. The model is also applied to pattern recognition. It becomes possible with a conventional model to restrict the category increase of memory patterns. © 2007 Wiley Periodicals, Inc. Electr Eng Jpn, 162(3): 51– 57, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.20320     

Synthesis and Biological Evaluation of Ferrocenylquinoline as a Potential Antileishmanial Agent

The emergence of resistance against antileishmanial drugs in current use necessitates the search for new classes of antileishmanial compounds. Herein we report the design, synthesis, and evaluation of a novel ferrocenylquinoline for activity against Leishmania donovani. 7‐Chloro‐N‐[2‐(1H‐5‐ferrocenyl‐1,2,3‐triazol‐1‐yl)ethyl]quinolin‐4‐amine ( 1 ) was generated by coupling an iron(II) ethynylferrocene species with 4‐(2‐ethylazido)amino‐7‐chloroquinoline using click chemistry. The synthesized compound 1 was tested for its antileishmanial activity using both promastigote and amastigote stages of L. donovani. Compound 1 showed promising anti‐promastigote activity, with an IC₅₀ value of 15.26 μM and no cytotoxicity toward host splenocytes. From the battery of tests conducted in this study, it appears that this compound induces parasite death by promoting oxidative stress and depolarizing the mitochondrial membrane potential, thereby triggering apoptosis. These results suggest that ferrocenylquinoline 1 is a suitable lead for the development of new antileishmanial drugs.